Project description:Papillomatous digital dermatitis (PDD) is a polymicrobial infection in soft tissue adjacent to the hoof and is the leading cause of lameness in dairy cattle. Treponema phagedenis-like (TPL) spirochetes are a constant feature of PDD lesions and are localized deep in infected tissue. Host-cell response mechanisms to TPL bacteria are poorly understood. To assess how bovine macrophages respond to cellular constituents of TPL spirochetes, changes in transcription were analyzed using serial analysis of gene expression (SAGE) and real time RT-PCR. This analysis revealed that bovine macrophages responded to stimulation with TPL-lysates by induction of several pro-apoptotic genes. Although several proinflammatory cytokines (eg. IL-6 and IL-8) are induced in treated macrophages, receptors and their accessory proteins for IL-1, IL-6, and IL-11 are either down regulated or unchanged. Surprisingly, IL-1beta is not induced and IL-18 is suppressed in treated macrophages. Two genes encoding proteins having negative effects on NF?B, I?B and Siva-1, are significantly induced in stimulated cells. Combined, these data suggest that the innate immune response of bovine macrophages exposed to TPL cellular constituents are impaired thereby enabling bacteria to resist clearance and induce lesion formation. Keywords: Bovine macrophage, transcription response, spirochete bacteria An immortalized bovine macrophage cell line (BoMac) was used throughout this study (supplied by Dr. Judith Stabel, USDA, ARS, NADC, Ames, IA). Cells were propagated in RPMI 1640 medium (GIBCO, Gaithersburg, MD) containing 10% fetal calf serum (Colorado Serum Company), 100µg/ml streptomycin, and 100U/ml penicillin (Sigma, St. Louis, MO) at 37C with 5% CO2. PDD spirochetes (Treponema phagedenis-like strain 1A) in the exponential phase of growth were harvested by centrifugation (10,000g for 15 min.) and washed twice with PBS. Cell lysates were prepared by sonication using short-wave pulses of 30s each (Vibra Cell, Sonics and Materials Inc, Danbury, Connecticut) until no intact cells were observed by dark field microscopy. Protein concentrations of whole cell sonicates (WCS) were determined using the Bradford assay (Bio-Rad, Hercules, CA) following the manufacturers instructions. Bovine macrophages grown to about 90% confluence were stimulated with WCS lysate of TPL strain 1A (1 µg/ml) suspended in complete RPMI 1040. Cells were incubated at 37oC for 4, 8, and 18 hrs. Cell monolayers were disrupted with RLT+ lysis solution and total cellular RNA isolated with RNeasy kits (Qiagen) according to the manufacturer’s instructions. Where appropriate cell suspensions were further disrupted by homogenization using Qiashredder columns (Qiagen). Residual DNA was removed by DNaseI treatment, followed by RNA purification (Qiagen). Cellular mRNA was enriched from total RNA using PolyATract mRNA isolation system II (Promega, Madison, WI) and used as starting material for preparation of SAGE libraries. Two SAGE libraries were prepared from 5 ug ds-cDNA, one each from non-stimulated and WCS-stimulated macrophages (4 hr treatment) following established protocols using NlaIII as the anchoring enzyme. Concatermerized ditags were size selected by electrophoresis through polyacrylamide gels, then ligated with SphI-digested pZERO-1 (Invitrogen Corp.) and used to transform E. coli to zeocin resistance. Transformed colonies were picked at random to 96-well plates and plasmid DNA prepared by alkaline lysis (Qiagen). Plasmid DNA was sequenced unidirectionally from the T7 primer region using fluorescent dideoxy chain-termination reactions separated in a 3700-sequence analyzer (Applied Biosystems, Inc.). Sequence data interpreted using Phred and Sequencher (Gene Codes Corporation, Ann Arbor, MI) were combined and edited manually. Inserts from a total of 2155 and 2131 plasmids were sequenced with average error rates of 0.00435 and 0.00977 from control and stimulated cell libraries, respectively. Data files for each library were processed with eSAGE software to extract ditags, remove duplicate sequences, and compare the two libraries to determine statistically significant differences in tag frequency. SAGE data were validated by quantifying transcription levels of selected genes using RNA isolated from three distinct biological replicates each of treated and control samples. Semiquantitative RT-PCR was done in a DNA Engine Opticon 2 detection system (MJ Research, Inc) using QuantiTect™ SYBR Green PCR Kit (Qiagen), gene specific primers (Table 1), with 200 ng/µl cDNA template. PCR primers were designed using Primer Design 5 software (Sci Ed Central) from sequences obtained from GenBank. All reactions were performed in triplicate and qRT-PCR data were analyzed by using the 2-(??Ct) method. The ?-actin gene was used as the control gene (?Ct) and non-stimulated samples were used as the calibrator (??Ct).

Project description:Proteins are the major constituents of muscle and are key molecules regulating the metabolic changes during conversion of muscle to meat. Brazil is one of the largest exporters of beef and most Brazilian cattle are composed by zebu (Nellore) genotype. Bos indicus beef is generally leaner and tougher than Bos taurus such as Angus. The aim of this study was to compare the muscle proteomic and phosphoproteomic profile of Angus and Nellore. Seven animals of each breed previously subjected the same growth management were confined for 84 days. Proteins were extracted from Longissimus lumborum samples collected immediately after slaughter and separated by two-dimensional electrophoresis. Pro-Q Diamond stain was used in phosphoproteomics. Proteins identification was performed using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Tropomyosin alpha-1 chain, troponin-T, myosin light chain-1 fragment, cytoplasmic malate dehydrogenase, alpha-enolase and 78 kDa glucose-regulated protein were more abundant in Nellore, while myosin light chain 3, prohibitin, mitochondrial stress-70 protein and heat shock 70 kDa protein 6 were more abundant in Angus (P<0.05). Nellore had higher phosphorylation of myosin regulatory light chain-2, alpha actin-1, triosephosphate isomerase and 14-3-3 protein epsilon. However, Angus had greater phosphorylation of phosphoglucomutase-1 and troponin-T (P<0.05). Therefore, proteins involved in contraction and muscle organization, myofilaments expressed in fast or slow-twitch fibers and heat shock proteins localized in mitochondria or sarcoplasmic reticulum and involved in cell flux of calcium and apoptosis might be associated with differences in beef quality between Angus and Nellore. Furthermore, prohibitin appears to be a potential biomarker of intramuscular fat in cattle. Additionally, differences in phosphorylation of myofilaments and glycolytic enzymes could be involved with differences in muscle contraction force, susceptibility to calpain, apoptosis and postmortem glycolysis, which might also be related to differences in beef quality among Angus and Nellore.

Project description:BackgroundReference genomes are essential in the analysis of genomic data. As the cost of sequencing decreases, multiple reference genomes are being produced within species to alleviate problems such as low mapping accuracy and reference allele bias in variant calling that can be associated with the alignment of divergent samples to a single reference individual. The latest reference sequence adopted by the scientific community for the analysis of cattle data is ARS_UCD1.2, built from the DNA of a Hereford cow (Bos taurus taurus-B. taurus). A complementary genome assembly, UOA_Brahman_1, was recently built to represent the other cattle subspecies (Bos taurus indicus-B. indicus) from a Brahman cow haplotype to further support analysis of B. indicus data. In this study, we aligned the sequence data of 15 B. taurus and B. indicus breeds to each of these references.ResultsThe alignment of B. taurus individuals against UOA_Brahman_1 detected up to five million more single-nucleotide variants (SNVs) compared to that against ARS_UCD1.2. Similarly, the alignment of B. indicus individuals against ARS_UCD1.2 resulted in one and a half million more SNVs than that against UOA_Brahman_1. The number of SNVs with nearly fixed alternative alleles also increased in the alignments with cross-subspecies. Interestingly, the alignment of B. taurus cattle against UOA_Brahman_1 revealed regions with a smaller than expected number of counts of SNVs with nearly fixed alternative alleles. Since B. taurus introgression represents on average 10% of the genome of Brahman cattle, we suggest that these regions comprise taurine DNA as opposed to indicine DNA in the UOA_Brahman_1 reference genome. Principal component and admixture analyses using genotypes inferred from this region support these taurine-introgressed loci. Overall, the flagged taurine segments represent 13.7% of the UOA_Brahman_1 assembly. The genes located within these segments were previously reported to be under positive selection in Brahman cattle, and include functional candidate genes implicated in feed efficiency, development and immunity.ConclusionsWe report a list of taurine segments that are in the UOA_Brahman_1 assembly, which will be useful for the interpretation of interesting genomic features (e.g., signatures of selection, runs of homozygosity, increased mutation rate, etc.) that could appear in future re-sequencing analysis of indicine cattle.

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Bos taurus

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